Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

Lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper enzymes discovered ∼10 years ago [13] and are now recognized to play major roles in the decomposition of recalcitrant biomass both in nature and in industrial contexts. Using an exogenous source of electrons [2,4], LPMOs use an active site copper ion to activate oxygen and oxidize polysaccharides [2,3,5] (Figure 1, top panel). Some recent reviews have focused on structure [6,7], mechanism [8,9], biological function [1012] or provided a broader perspective [13,14]. This review aims to briefly introduce this very fast moving field and gives an update covering primarily the last 2–3 years.

Basic LPMO reaction scheme.

Figure 1.
Basic LPMO reaction scheme.

Top: the LPMO reaction is initiated with the 1e reduction of Cu(II) (yellow sphere) to Cu(I) (blue sphere). From here, several paths may be taken, depending on the conditions after Cu reduction, and both monooxygenase (a) and peroxygenase (b) reactions have been demonstrated. LPMOs are known to degrade different polysaccharides, primarily with α-1,4 or β-1,4-linked glucose backbone or chitin. If suitable substrate (here exemplified by cellulose) and oxygen co-substrate are present, the substrate may be oxidized at either the C1 or C4 carbons (marked) depending on the specific LPMO and returns the LPMO to its Cu(II) resting state (this has been referred to as the canonical or coupled reaction [15,16]). If the reduced LPMO in the Cu(I) state is in the presence of H2O2, this co-substrate will react fast to hydroxylate the substrate while retaining the LPMO in the Cu(I) state (uncoupled reaction in ref. [16]). A similar path may be involved in the presence of substrates that do not displace the axial copper ligand (non-canonical pathway). In the absence of substrate and H2O2 co-substrate, the reduced LPMO reacts with O2 leading to formation of H2O2 (c, referred often to as futile cycle). The LPMO is hereby returned to the resting Cu(II) state. Bottom: likely steps in the monooxygenase reaction with O2 as a co-substrate. (A) The Cu(II) resting state. Electron transfer from an external reductant produces the Cu(I) state (B). The addition of molecular oxygen, 2H+ and an additional electron in the presence of suitable substrate leads to formation of a reactive oxygen species (here intentionally not specified and indicated as Ox) and eventually the oxidized product and water (F). Hydrogen abstraction and subsequent hydroxylation via an oxygen-rebound mechanism forms intermediates C and D. As the details of electron transfer and active oxygen species are not yet verified, the reaction is correctly balanced going from B to F, but not in the shaded area. From (F), the final product is formed by spontaneous elimination in water.

Figure 1.
Basic LPMO reaction scheme.

Top: the LPMO reaction is initiated with the 1e reduction of Cu(II) (yellow sphere) to Cu(I) (blue sphere). From here, several paths may be taken, depending on the conditions after Cu reduction, and both monooxygenase (a) and peroxygenase (b) reactions have been demonstrated. LPMOs are known to degrade different polysaccharides, primarily with α-1,4 or β-1,4-linked glucose backbone or chitin. If suitable substrate (here exemplified by cellulose) and oxygen co-substrate are present, the substrate may be oxidized at either the C1 or C4 carbons (marked) depending on the specific LPMO and returns the LPMO to its Cu(II) resting state (this has been referred to as the canonical or coupled reaction [15,16]). If the reduced LPMO in the Cu(I) state is in the presence of H2O2, this co-substrate will react fast to hydroxylate the substrate while retaining the LPMO in the Cu(I) state (uncoupled reaction in ref. [16]). A similar path may be involved in the presence of substrates that do not displace the axial copper ligand (non-canonical pathway). In the absence of substrate and H2O2 co-substrate, the reduced LPMO reacts with O2 leading to formation of H2O2 (c, referred often to as futile cycle). The LPMO is hereby returned to the resting Cu(II) state. Bottom: likely steps in the monooxygenase reaction with O2 as a co-substrate. (A) The Cu(II) resting state. Electron transfer from an external reductant produces the Cu(I) state (B). The addition of molecular oxygen, 2H+ and an additional electron in the presence of suitable substrate leads to formation of a reactive oxygen species (here intentionally not specified and indicated as Ox) and eventually the oxidized product and water (F). Hydrogen abstraction and subsequent hydroxylation via an oxygen-rebound mechanism forms intermediates C and D. As the details of electron transfer and active oxygen species are not yet verified, the reaction is correctly balanced going from B to F, but not in the shaded area. From (F), the final product is formed by spontaneous elimination in water.

Classification and new families

LPMOs (recently assigned EC 1.14.99.53-56) are classified in the Carbohydrate Active enZymes database (CAZy, http://www.cazy.org) [17] as Auxiliary Activities (AAs, redox enzymes involved in carbohydrate degradation) [18], within families AA9, AA10, AA11, AA13, AA14 and AA15. Sequences that could define new families have been identified in genomic data (e.g. in ref. [19]), but biochemical evidence is needed to corroborate that they are LPMOs. AA9–AA13 have been known for some years and represent bacterial (AA10), viral (AA10) and fungal LPMOs (AA9, AA11 and AA13) acting on β-1,4-linked (AA9, AA10, AA11) or α-1,4-linked (AA13) polysaccharides. Regiospecificity is not strictly associated with families and can be substrate-dependent [15,20].

AA14 has recently been defined based on the characterization of two enzymes, PcAA14_A and PcAA14_B, from the fungus Pycnoporus coccineus [21]. Phylogenetic analysis showed that the newly identified AA14 sequences strongly cluster together distant from other fungal LPMO families. PcAA14s act synergistically with Trichoderma reesei enzyme cocktails during saccharification of pretreated wood. C1-oxidised xylotriose was detected as a degradation product from PcAA14_A in combination with a GH11 (glycoside hydrolase 11) xylanase on birchwood cellulosic fibers. No enzyme activity was detected on 11 isolated polysaccharides including cellulose and birchwood xylan.

The range of LPMO-producing organisms was greatly expanded with the discovery of AA15 members, which are the first of animal origin [22]. While the first isolated AA15 from the cellulose-degrading insect firebrat acted on cellulose, chitin-degrading activity of AA15 LPMOs was also documented and could explain the presence of AA15 in organisms such as Drosophila where it may serve during re-modelling of endogenous chitin during development and metamorphosis.

The LPMO reaction: mechanisms and assays

The catalytic mechanism of LPMOs has been the centre of considerable scientific debate. Monooxygenase activity was first proposed in 2010 [2], and the mononuclear copper active site was shown in 2011 [3]. The variation in geometry of Cu coordination in LPMOs has recently been extensively reviewed [9] and not discussed in depth here. Likely events during the catalytic cycle are hydrogen abstraction at C1 or C4 of the substrate, followed by hydroxylation through an oxygen-rebound mechanism and elimination to the final product (Figure 1). Polysaccharide oxidation is accompanied by reduction of one of the atoms in molecular oxygen to water. Hydrogen abstraction from polysaccharides requires formation of a very powerful oxidative species, and both reaction and active site geometry bear resemblance to the oxidation of methane to methanol by particulate methane monooxygenase (pMMO) [3,23].

In LPMOs, the activation of oxygen is accomplished by reduction of Cu(II) to Cu(I) and relocation of the electron to dioxygen upon binding to Cu(I)-LPMO [5]. Several possible active oxygen species have been discussed [8,24], as has the order of substrate and co-substrate binding and generation of intermediate species [25,26], with recent theoretical studies mostly in favour for equatorial Cu-oxo or Cu-oxyl species [2628]. Electrons for the reaction are provided by exogenous donors that can be small molecules or proteins, as comprehensively described [4], and additional ones described since, e.g. [2931]. One conundrum has been how electrons are delivered, since protein electron donors seem to bind at the same site as the substrate [32].

A stable reactive LPMO system is achieved only when the supply of electrons matches the concentration of the enzyme and the concentration of productive binding sites on the polymeric substrate [33]. In the canonical LPMO mechanism (the coupled pathway in ref. [34], where the authors propose a random-sequential kinetic mechanism), the polymeric substrate-binding mode blocks the axial position on the copper and the enzyme proceeds through the catalytic cycle without release of the activated oxygen [35]. In the absence of substrate though, LPMOs (in computational studies) release protonated superoxide [5], which can spontaneously convert into H2O2. Production of hydrogen peroxide was first shown experimentally in ref. [36], and later by many others. Hydroxyl radicals may be formed if hydrogen peroxide binds to the reduced enzyme (Cu(I)-LPMO) [16,28,34] and could participate in alternative mechanisms in a ‘caged’ [28] or free form [16]. Such reactivity is supported by work on small molecule mimics [3739]. Polymeric substrates that bind differently to the surface of LPMOs and are unable to block the axial position may be cleaved through a related reaction mechanism [15]. Experiments with small molecule mimics have also recently suggested the involvement of a Cu(III)-hydroperoxo intermediate in the LPMO reaction cycle [40].

The reaction requires tight control in biological systems to prevent detrimental oxidation of vital secreted enzymes including the LPMO itself (as shown, for example, in ref. [41]), or the outer envelope of the organism. It has been proposed that H2O2 is the preferred co-substrate for LPMOs, in a peroxygenase reaction where a single priming reduction to Cu(I) is needed [41] (Figure 1). Theoretical calculations support feasibility of this reaction [27,28], but it is restricted by fast inactivation rates of the LPMO [16,34,42]. It has been shown that removal of H2O2 by catalase is useful in lignocellulosic saccharification slurries and that LPMO-rich cellulolytic enzyme cocktails are more sensitive to added H2O2 when the level of dissolved oxygen is low [43]. It has also been shown that H2O2 reacts with components in lignocellulosic slurries leading to acidification and production of CO2 [44]. This observation demonstrates that it is very unlikely for H2O2 to react quantitatively and productively with LPMOs during saccharification of lignocellulose, as has been postulated by some [45]. The biological relevance of H2O2 as a co-substrate was also probed by incubation of the Neurospora crassa secretome with cellulose. The addition of peroxidase to remove the proposed co-substrate H2O2 did not have any effect [16]. Moreover, it has been shown that the coprophilous fungus Podospora anserina has a strict requirement for catalase activity for growth on complex biomass where LPMOs will be involved in the saccharification of the substrate [46]. More work is required to understand the relative importance of alternative LPMO pathways in different biological and industrial contexts.

A recent excellent enzyme kinetics study [34] combines several assay methods which enable an accurate determination of catalytic constants, but a single approach may be sufficient in specific cases. LPMO activity assays can be grouped according to the parameter that is monitored and quantified: (1) consumption of the co-substrate dioxygen [4749]; (2) release of oxidized sugars [5053] or oligomers [15,35]; (3) synergistic effect when combined with hydrolases [21,43,54]; (4) labelling of oxidized surfaces of insoluble substrates [55]; (5) production of H2O2 in the absence of polymeric substrate [56]; (6) reaction with H2O2 in the absence of polymeric substrate [57]; (7) generation of carboxylate moieties (for C1-oxidizing LPMOs) [58]. Caveats such as the reactivity of any free copper in the reaction mixtures [3], or the buffer constituents [57], must be always kept in mind.

Recent structural studies

Structural studies, reviewed, for example, in refs [6,7,13], have added much to our understanding of LPMOs. Table 1 updates the overview of known 3D structures in Frandsen and Lo Leggio [6] (a full table of all structures can be found in https://sid.erda.dk/sharelink/HB3w2p0k7s).

Table 1
Structures of LPMO published since Frandsen and Lo Leggio [6]
CAZy Family Organism Protein name PDB code ASU Crystallization and diffraction data Active site Specificity 
Protein concentration and buffer Crystallization conditions Space group Resolution (Å) Element (oxidation) Residues Substrates Site of attack Comments References 
AA9 Aspergillus fumigatus PMO-5
AfAA9_B
Afum_AFUA_4G07850
Afu4g07850 
5X6A N/R 0.2 M MgCl2, 25% (w/v) PEG3350
0.1 M Bis–Tris
pH 6.0 
P1.70 – His1
His86
Tyr175 
N/D N/D Disordered active site His1. No active site metal To be published 
AA9 Collariella virescens CvAA9_A 5NLT 6.3 mg/ml
0.02 M Na-acetate pH 5.5 
1.6 M (NH4)2SO4
0.1 M NaCl
0.1 M HEPES
pH 7.5 
P1.95 Cu(I) meHis1
His79
Tyr169 
PASC
Cello-oligo-saccharides
Xyloglucan
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate [15
AA9 Heterobasidion irregulare HiAA9_B
HiLPMO9B 
5NNS 10 mg/ml
0.02 M HEPES
pH 7.5 (0.15 M NaCl) 
22% (w/v) Na-polyacrylic acid, 0.02 M MgCl2
0.1 M HEPES
pH 7.5 
P21 2.10 Cu His1
His80
Tyr166 
PASC C1 PDB entry on hold [59
AA9 Lentinus
similis 
LsAA9_A 5N04 8.5 mg/ml
0.02 M Na-acetate pH 5.5 
3.0 M NaCl
0.1 M Citric acid
pH 3.5 
P4132 1.76 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Disordered active site His78, partially replaced by a chloride ion 
[60
AA9 Lentinus similis LsAA9_A 5N05 8.5 mg/ml
0.02 M Na-acetate pH 5.5 
3.4 M NaCl
0.1 M Citric acid
pH 3.5 
P4132 1.58 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Cellohexaose bound in the active site 
[60
AA9 Lentinus similis LsAA9_A 5NKW 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
3.3 M NaCl
0.1 M Citric acid
pH 3.5 (soaked in pH 5.5) 
P4132 1.48 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1,3;1,4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Glucomannan oligosaccharide in the active site.
Oligosaccharides modelled near Gln14 
[15
AA9 Lentinus similis LsAA9_A 5NLN 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
3.6 M NaCl
0.1 M citric acid
pH 4.5 (soaked in pH 5.5) 
P4132 1.90 Cu(II) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Xylopentaose oligosaccharide in the active site.
The active site axial position is partially occupied by an exogenous ligand (Cl) mimicking an oxygen species 
[15
AA9 Lentinus similis LsAA9_A 5NLO 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
3.6 M NaCl
0.1 M citric acid
pH 4.5 (soaked in pH 5.5)

 
P4132 1.33 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Xylopentaose oligosaccharide modelled in the active site. 
[15
AA9 Lentinus similis LsAA9_A 5NLP 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
4.1 M NaCl
0.1 M citric acid
pH 4.0 (soaked pH 5.5) 
P4132 1.59 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Soaked with Xylotetraose
Xylo-oligosaccharides modelled in the active site and near Gln14 and Tyr21 
[15
AA9 Lentinus similis LsAA9_A 5NLQ 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
4.4 M NaCl
0.1 M citric acid
pH 4.5 (soaked in pH 5.5) 
P4132 1.50 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Soaked with Xylotriose
Xylo-oligosaccharides modelled in the active site and near Asp38 and the glycosylation (Asn33) 
[15
AA9 Lentinus similis LsAA9_A 5NLR 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
3.5 M NaCl
0.1 M citric acid, pH 3.5 
P4132 2.00 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Soaked with 1,3:1,4-β-glucotetraose.
Partially overlapping β(1,4)-gluco- oligosaccharides modelled in the active site 
[15
AA9 Lentinus similis LsAA9_A 5NLS 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
3.2 M NaCl
0.1 M citric acid, pH 3.5 (soaked in pH 5.5) 
P4132 1.75 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Cellopentaose modelled in the active site 
[15
AA9 Neurospora crassa NcAA9_D
NcLPMO9D
PMO-2
NCU01050
GH61-4 
5TKF 12.6 mg/ml
0.02 M Na-acetate pH 5.0 
22% (w/v) PEG3350
0.1 M HEPES
pH 6.4 
P2.10 Cu(I) meH1
His84
Tyr168 
PASC C4 In chain B and D O2 molecules are modelled near the active site interacting with His157 and Gln166 [61
AA9 Neurospora crassa NcAA9_D
NcLPMO9D
PMO-2
NCU01050
GH61-4 
5TKG 12.5 mg/ml
0.02 M Na-acetate pH 5.0 
25% (w/v) PEG3350
0.1 M HEPES
pH 6.0 
P21 1.20 Cu(II) meH1
His84
Tyr168 
PASC C4 Additional details on crystallzation in ref. [61[62
AA9 Neurospora crassa NcAA9_D
NcLPMO9D
PMO-2
NCU01050
GH61-4 
5TKH N/R 25% (w/v) PEG3350
0.1 M HEPES
pH 6.0 
P21 1.20 Cu(II)/Cu(I) meH1
His84
Tyr168 
PASC C4 Proposed oxygen species modelled in the active site equatorial position interacting with His157 and Gln166 [62
AA9 Neurospora crassa NcAA9_D
NcLPMO9D
PMO-2
NCU01050
GH61-4 
5TKI (X-ray) 12.2 mg/ml
0.02 M Na-acetate pH 5.0 
25% (w/v) PEG3350
0.09 M HEPES
pH 6.0 
P21 1.50 Cu(II) meH1
His84
Tyr168 
PASC C4 Room temperature X-ray data collection
Joint X-ray and neutron crystal structure
Additional crystallzation conditions in ref. [61
[62
AA9 Neurospora crassa NcAA9_D
NcLPMO9D
PMO-2
NCU01050
GH61-4 
5TKI (neutron) 12.2 mg/ml
0.02 M Na-acetate pH 5.0 
25% (w/v) PEG3350
0.09 M HEPES
pH 6.0 
P21 2.12 Cu meH1
His84
Tyr168 
PASC C4 Joint X-ray and neutron crystal structure
Additional crystallzation conditions in ref. [61
[62
AA9 Neurospora crassa NcAA9_A
NcLPMO9A
NCU02240
GH61-1 
5FOH – 0.2 M LiSO4
20% (w/v)
PEG3350
pH 6.5 
P3221 1.60 Cu(I) His1
His81
Tyr164 
Avicel N/D Activity in ref [63To be published 
AA9 Thermothelomyces
thermophila 
MtAA9_D
MtPMO3
MYCTH_92668 
5UFV 20 mg/ml

 
18% (w/v)
PEG 6000
0.1 M Na-citrate
pH 3.9 
P21 2.45 Cu(I) MeHis1
His75
Tyr169 
PASC C1 Reconstituted with excess CuSO4 at pH 5.0 for 4 h [47
AA9 Trichoderma reesei EGIV
Egl4
EG4
LPMO4
HjAA9_A
HjLPMO9A
TrAA9_A
TrCel61A 
5O2W 30 mg/ml
0.025 M
Tris–HCl
pH 7.5
0.025 M NaCl 
1.6 M (NH4)2SO4
0.1 M citric acid
pH 4.0 
P21 1.78 Cu(I) MeHis1
His86
Tyr174 
PASC C1/C4 Structure shows that a predicted linker is an integral part of the catalytic domain [64
AA9 Trichoderma reesei EGIV
Egl4
EG4
LPMO4
HjAA9_A
HjLPMO9A
TrAA9_A
TrCel61A 
5O2X 30 mg/ml
0.025 M
Tris–HCl
pH 7.5
0.025 M NaCl 
1.6 M (NH4)2SO4
0.1 M citric acid
pH 4.0 
P21 0.95 Cu(II) MeHis1
His86
Tyr174 
PASC C1/C4 Structure shows that a predicted linker is an integral part of the catalytic domain [64
AA10 Bacillus amylolique-faciens BaAA10_A
BaCBM33
ChbB
Rbam17540 
5IJU 4.7 mg/ml
0.02 M Na-acetate pH 5.0
0.25 M NaCl 
20% (w/v)
PEG6000
0.1 M acetate pH 5.0
0.2 M CaCl2 (+seed stock) 
P21 1.70 Cu(II) His28
His125
Phe196 
β-chitin C1 Microseeded using crystals grown in 0.1 M MMT pH 4.0, 25% (w/v) PEG1500.
CuCl2 added in stoichiometric amounts C1 oxidation of β-chitin reported 
[65
AA10 Bacillus licheniformis BlLPMO10A
BlAA10_A 
5LW4 (NMR) 0.8 mM BlLPMO10A 0.025 M Na-phosphate
pH 5.0
0.01 M NaCl (90%/10% H2O/D2O) 
– – – – His1
His90
Phe161 
 – NMR structure 20 conformers
No active site metal
Active site disorder reminiscent of AoAA13 (PDB 5T7K) 
To be published 
AA10 Bacillus thuringiensis- serovar BtAA10_A
LPMO10A
BtLPMO10A 
5WSZ – 0.2 M Mg-acetate
0.1 M Na-cacodylate
pH 6.5, 20% (w/v) PEG8000 
P21212.57 Cu(I) His1
His85
Phe158 
– – – To be published 
AA10 Jonesia denitrificans JdAA10_A JdLPMO10A
Jden_1381 
5VG0 (X-ray) 48 mg/ml
20 mM Tris–HCl pH 8.0 
1.9 M dl-malic acid pH 7.0 P212121 1.10 Cu(II) His32
His109
Phe164 
α-chitin
β-chitin 
C1 Incubated with a threefold molar excess of CuSO4 for 30 min at room temperature
Proposed peroxide ion in equatorial position 
[66
AA10 Jonesia denitrificans JdAA10_A JdLPMO10A
Jden_1381 
5VG1 (neutron) 48 mg/ml
20 mM Tris–HCl pH 8.0 
1.9 M dl-malic acid pH 7.0 P212121 2.10 Cu(II) His32
His109
Phe164 
α-chitin
β-chitin 
C1 Incubated with a threefold molar excess of CuSO4 for 30 min at room temperature
Proposed peroxide ion in equatorial position 
[66
AA10 Micromonospora aurantiaca MaAA10_B
MaLPMO10B 
5OPF 21.9 mg/ml, 0.02 M Bis–Tris pH 6.0 0.04 M potassium phosphate
16% (w/v) PEG8000
20% (v/v) glycerol 
P212121 1.08 Cu(II) His37
His144
Phe221 
PASC
β-chitin 
C1/C4 The C-terminal G230 from a symmetry related molecule occupy the ligand positions on the active site Cu, albeit with distorted geometry.
Authors claim Cu(I) 
[67
AA10 Thermobifida fusca TfAA10_A TfLPMO10A
E7
Tfu_1268 
5UIZ 8.5 mg/ml
0.1 M HEPES
pH 7.5
0.1 M NaCl
5% Glycerol
5% Ethylene-glycol 
0.1 M HEPES
pH 7.5
4.3 M NaCl
– or –0.1 M Na-acetate
pH 4.6
20% (v/v)
2-propanol
0.2 M CaCl 
P3221 2.00 Cu(I)
A 0.5
B 0.31 
His37
His144
Tyr213 
PASC
Avicel
β-chitin 
C1/C4 (C1 on chitin) Regiospecificity in ref. [68]
Conflicting information on crystallization in publication and PDB deposition entry 
[69
AA13 Aspergillus oryzae AoAA13
AO090701000246
AOR_1_454114 
5T7J 3 mg/ml
0.02 M MES
pH 6.0
0.125 M NaCl 
0.02 M Zn-acetate
0.1 M Malate/MES/Tris pH 5.0
20% (w/v)
PEG3000 
P212121 1.65 Zn(II) MeHis1
His91
Tyr224 
N/D (starch) N/D (C1) Activity inferred from refs [70,71[72
AA13 Aspergillus oryzae AoAA13
AO090701000246
AOR_1_454114 
5T7N 3 mg/ml
0.02 M MES
pH 6.0
0.125 M NaCl 
0.02 M Zn-acetate
0.1 M Malate/MES/Tris pH 5.0
20% (w/v)
PEG3000 
P212121 1.60 Zn(II)
0.7 
MeHis1
His91
Tyr224 
N/D
(starch) 
N/D (C1) Glucosyl-maltotriose bound outside the active site.
Activity inferred from refs [70,71
[72
AA13 Aspergillus oryzae AoAA13
AO090701000246
AOR_1_454114 
5T7K 3 mg/ml
0.02 M MES
pH 6.0
0.125 M NaCl 
0.02 M Zn-acetate
0.1 M Malate/MES/Tris pH 5.0
20% (w/v)
PEG3000 
P212121 1.30 Zn(II)
0.2 
MeHis1
His91
Tyr224 
N/D (starch) N/D (C1) His91 flipped out of active site.
Stacking with Phe166.
Activity inferred from refs [70,71
[72
AA13 Aspergillus oryzae AoAA13
AO090701000246
AOR_1_454114 
5LSV 3 mg/ml
0.02 M MES
pH 6.0
0.125 M NaCl 
0.02 M Zn-acetate
0.1 M Malate/MES/Tris pH 5.0
20% (w/v)
PEG3000 
P212121 1.10 Zn(II)
A 0.8
B 0.2 
MeHis1
His91
Tyr224 
N/D (starch) N/D (C1) Maltose bound outside the active site.
Activity inferred from refs [70,71
[72
AA14 Pycnoporus coccineus PcAA14_B
PcAA14B 
5NO7 28 mg/ml
0.05 M Na-acetate pH 5.2 
2.4 M (NH4)2SO4 0.1 M citric acid
pH 4.4 
P422121 3.01 – His1
His99
Tyr176 
Xylan (cellulose-associated) C1 C1-oxidized xylo-oligosaccharides released after synergistic action with GH11 xylanases [21
AA15 Thermobia domestica TdAA15_A
TdAA15A 
5MSZ 28 mg/ml
0.05 M Na-acetate pH 5.2 
0.1 M Na-citrate
pH 5.5
0.1 M LiCl, 10–25% (w/v) PEG6000 
P22121 1.10 Cu(I) His1
His91
Tyr184 
Avicel β-chitin C1 First structure of an LPMO belonging to the phylum of Arthropoda [22
CAZy Family Organism Protein name PDB code ASU Crystallization and diffraction data Active site Specificity 
Protein concentration and buffer Crystallization conditions Space group Resolution (Å) Element (oxidation) Residues Substrates Site of attack Comments References 
AA9 Aspergillus fumigatus PMO-5
AfAA9_B
Afum_AFUA_4G07850
Afu4g07850 
5X6A N/R 0.2 M MgCl2, 25% (w/v) PEG3350
0.1 M Bis–Tris
pH 6.0 
P1.70 – His1
His86
Tyr175 
N/D N/D Disordered active site His1. No active site metal To be published 
AA9 Collariella virescens CvAA9_A 5NLT 6.3 mg/ml
0.02 M Na-acetate pH 5.5 
1.6 M (NH4)2SO4
0.1 M NaCl
0.1 M HEPES
pH 7.5 
P1.95 Cu(I) meHis1
His79
Tyr169 
PASC
Cello-oligo-saccharides
Xyloglucan
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate [15
AA9 Heterobasidion irregulare HiAA9_B
HiLPMO9B 
5NNS 10 mg/ml
0.02 M HEPES
pH 7.5 (0.15 M NaCl) 
22% (w/v) Na-polyacrylic acid, 0.02 M MgCl2
0.1 M HEPES
pH 7.5 
P21 2.10 Cu His1
His80
Tyr166 
PASC C1 PDB entry on hold [59
AA9 Lentinus
similis 
LsAA9_A 5N04 8.5 mg/ml
0.02 M Na-acetate pH 5.5 
3.0 M NaCl
0.1 M Citric acid
pH 3.5 
P4132 1.76 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Disordered active site His78, partially replaced by a chloride ion 
[60
AA9 Lentinus similis LsAA9_A 5N05 8.5 mg/ml
0.02 M Na-acetate pH 5.5 
3.4 M NaCl
0.1 M Citric acid
pH 3.5 
P4132 1.58 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Cellohexaose bound in the active site 
[60
AA9 Lentinus similis LsAA9_A 5NKW 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
3.3 M NaCl
0.1 M Citric acid
pH 3.5 (soaked in pH 5.5) 
P4132 1.48 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1,3;1,4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Glucomannan oligosaccharide in the active site.
Oligosaccharides modelled near Gln14 
[15
AA9 Lentinus similis LsAA9_A 5NLN 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
3.6 M NaCl
0.1 M citric acid
pH 4.5 (soaked in pH 5.5) 
P4132 1.90 Cu(II) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Xylopentaose oligosaccharide in the active site.
The active site axial position is partially occupied by an exogenous ligand (Cl) mimicking an oxygen species 
[15
AA9 Lentinus similis LsAA9_A 5NLO 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
3.6 M NaCl
0.1 M citric acid
pH 4.5 (soaked in pH 5.5)

 
P4132 1.33 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Xylopentaose oligosaccharide modelled in the active site. 
[15
AA9 Lentinus similis LsAA9_A 5NLP 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
4.1 M NaCl
0.1 M citric acid
pH 4.0 (soaked pH 5.5) 
P4132 1.59 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Soaked with Xylotetraose
Xylo-oligosaccharides modelled in the active site and near Gln14 and Tyr21 
[15
AA9 Lentinus similis LsAA9_A 5NLQ 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
4.4 M NaCl
0.1 M citric acid
pH 4.5 (soaked in pH 5.5) 
P4132 1.50 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Soaked with Xylotriose
Xylo-oligosaccharides modelled in the active site and near Asp38 and the glycosylation (Asn33) 
[15
AA9 Lentinus similis LsAA9_A 5NLR 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
3.5 M NaCl
0.1 M citric acid, pH 3.5 
P4132 2.00 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Soaked with 1,3:1,4-β-glucotetraose.
Partially overlapping β(1,4)-gluco- oligosaccharides modelled in the active site 
[15
AA9 Lentinus similis LsAA9_A 5NLS 19.2 mg/ml
0.02 M Na-acetate pH 5.5 
3.2 M NaCl
0.1 M citric acid, pH 3.5 (soaked in pH 5.5) 
P4132 1.75 Cu(I) meHis1
His78
Tyr164 
PASC
Cello-oligosaccharides
Xyloglucan
Xylan
Xylo-oligosaccharides
Glucomannan
Mixed-linkage (1;3,1;4)-β-d-glucans 
C4 (C1 products also detected from polysaccharides) Preincubated with 1 mM Cu(II)acetate
Cellopentaose modelled in the active site 
[15
AA9 Neurospora crassa NcAA9_D
NcLPMO9D
PMO-2
NCU01050
GH61-4 
5TKF 12.6 mg/ml
0.02 M Na-acetate pH 5.0 
22% (w/v) PEG3350
0.1 M HEPES
pH 6.4 
P2.10 Cu(I) meH1
His84
Tyr168 
PASC C4 In chain B and D O2 molecules are modelled near the active site interacting with His157 and Gln166 [61
AA9 Neurospora crassa NcAA9_D
NcLPMO9D
PMO-2
NCU01050
GH61-4 
5TKG 12.5 mg/ml
0.02 M Na-acetate pH 5.0 
25% (w/v) PEG3350
0.1 M HEPES
pH 6.0 
P21 1.20 Cu(II) meH1
His84
Tyr168 
PASC C4 Additional details on crystallzation in ref. [61[62
AA9 Neurospora crassa NcAA9_D
NcLPMO9D
PMO-2
NCU01050
GH61-4 
5TKH N/R 25% (w/v) PEG3350
0.1 M HEPES
pH 6.0 
P21 1.20 Cu(II)/Cu(I) meH1
His84
Tyr168 
PASC C4 Proposed oxygen species modelled in the active site equatorial position interacting with His157 and Gln166 [62
AA9 Neurospora crassa NcAA9_D
NcLPMO9D
PMO-2
NCU01050
GH61-4 
5TKI (X-ray) 12.2 mg/ml
0.02 M Na-acetate pH 5.0 
25% (w/v) PEG3350
0.09 M HEPES
pH 6.0 
P21 1.50 Cu(II) meH1
His84
Tyr168 
PASC C4 Room temperature X-ray data collection
Joint X-ray and neutron crystal structure
Additional crystallzation conditions in ref. [61
[62
AA9 Neurospora crassa NcAA9_D
NcLPMO9D
PMO-2
NCU01050
GH61-4 
5TKI (neutron) 12.2 mg/ml
0.02 M Na-acetate pH 5.0 
25% (w/v) PEG3350
0.09 M HEPES
pH 6.0 
P21 2.12 Cu meH1
His84
Tyr168 
PASC C4 Joint X-ray and neutron crystal structure
Additional crystallzation conditions in ref. [61
[62
AA9 Neurospora crassa NcAA9_A
NcLPMO9A
NCU02240
GH61-1 
5FOH – 0.2 M LiSO4
20% (w/v)
PEG3350
pH 6.5 
P3221 1.60 Cu(I) His1
His81
Tyr164 
Avicel N/D Activity in ref [63To be published 
AA9 Thermothelomyces
thermophila 
MtAA9_D
MtPMO3
MYCTH_92668 
5UFV 20 mg/ml

 
18% (w/v)
PEG 6000
0.1 M Na-citrate
pH 3.9 
P21 2.45 Cu(I) MeHis1
His75
Tyr169 
PASC C1 Reconstituted with excess CuSO4 at pH 5.0 for 4 h [47
AA9 Trichoderma reesei EGIV
Egl4
EG4
LPMO4
HjAA9_A
HjLPMO9A
TrAA9_A
TrCel61A 
5O2W 30 mg/ml
0.025 M
Tris–HCl
pH 7.5
0.025 M NaCl 
1.6 M (NH4)2SO4
0.1 M citric acid
pH 4.0 
P21 1.78 Cu(I) MeHis1
His86
Tyr174 
PASC C1/C4 Structure shows that a predicted linker is an integral part of the catalytic domain [64
AA9 Trichoderma reesei EGIV
Egl4
EG4
LPMO4
HjAA9_A
HjLPMO9A
TrAA9_A
TrCel61A 
5O2X 30 mg/ml
0.025 M
Tris–HCl
pH 7.5
0.025 M NaCl 
1.6 M (NH4)2SO4
0.1 M citric acid
pH 4.0 
P21 0.95 Cu(II) MeHis1
His86
Tyr174 
PASC C1/C4 Structure shows that a predicted linker is an integral part of the catalytic domain [64
AA10 Bacillus amylolique-faciens BaAA10_A
BaCBM33
ChbB
Rbam17540 
5IJU 4.7 mg/ml
0.02 M Na-acetate pH 5.0
0.25 M NaCl 
20% (w/v)
PEG6000
0.1 M acetate pH 5.0
0.2 M CaCl2 (+seed stock) 
P21 1.70 Cu(II) His28
His125
Phe196 
β-chitin C1 Microseeded using crystals grown in 0.1 M MMT pH 4.0, 25% (w/v) PEG1500.
CuCl2 added in stoichiometric amounts C1 oxidation of β-chitin reported 
[65
AA10 Bacillus licheniformis BlLPMO10A
BlAA10_A 
5LW4 (NMR) 0.8 mM BlLPMO10A 0.025 M Na-phosphate
pH 5.0
0.01 M NaCl (90%/10% H2O/D2O) 
– – – – His1
His90
Phe161 
 – NMR structure 20 conformers
No active site metal
Active site disorder reminiscent of AoAA13 (PDB 5T7K) 
To be published 
AA10 Bacillus thuringiensis- serovar BtAA10_A
LPMO10A
BtLPMO10A 
5WSZ – 0.2 M Mg-acetate
0.1 M Na-cacodylate
pH 6.5, 20% (w/v) PEG8000 
P21212.57 Cu(I) His1
His85
Phe158 
– – – To be published 
AA10 Jonesia denitrificans JdAA10_A JdLPMO10A
Jden_1381 
5VG0 (X-ray) 48 mg/ml
20 mM Tris–HCl pH 8.0 
1.9 M dl-malic acid pH 7.0 P212121 1.10 Cu(II) His32
His109
Phe164 
α-chitin
β-chitin 
C1 Incubated with a threefold molar excess of CuSO4 for 30 min at room temperature
Proposed peroxide ion in equatorial position 
[66
AA10 Jonesia denitrificans JdAA10_A JdLPMO10A
Jden_1381 
5VG1 (neutron) 48 mg/ml
20 mM Tris–HCl pH 8.0 
1.9 M dl-malic acid pH 7.0 P212121 2.10 Cu(II) His32
His109
Phe164 
α-chitin
β-chitin 
C1 Incubated with a threefold molar excess of CuSO4 for 30 min at room temperature
Proposed peroxide ion in equatorial position 
[66
AA10 Micromonospora aurantiaca MaAA10_B
MaLPMO10B 
5OPF 21.9 mg/ml, 0.02 M Bis–Tris pH 6.0 0.04 M potassium phosphate
16% (w/v) PEG8000
20% (v/v) glycerol 
P212121 1.08 Cu(II) His37
His144
Phe221 
PASC
β-chitin 
C1/C4 The C-terminal G230 from a symmetry related molecule occupy the ligand positions on the active site Cu, albeit with distorted geometry.
Authors claim Cu(I) 
[67
AA10 Thermobifida fusca TfAA10_A TfLPMO10A
E7
Tfu_1268 
5UIZ 8.5 mg/ml
0.1 M HEPES
pH 7.5
0.1 M NaCl
5% Glycerol
5% Ethylene-glycol 
0.1 M HEPES
pH 7.5
4.3 M NaCl
– or –0.1 M Na-acetate
pH 4.6
20% (v/v)
2-propanol
0.2 M CaCl 
P3221 2.00 Cu(I)
A 0.5
B 0.31 
His37
His144
Tyr213 
PASC
Avicel
β-chitin 
C1/C4 (C1 on chitin) Regiospecificity in ref. [68]
Conflicting information on crystallization in publication and PDB deposition entry 
[69
AA13 Aspergillus oryzae AoAA13
AO090701000246
AOR_1_454114 
5T7J 3 mg/ml
0.02 M MES
pH 6.0
0.125 M NaCl 
0.02 M Zn-acetate
0.1 M Malate/MES/Tris pH 5.0
20% (w/v)
PEG3000 
P212121 1.65 Zn(II) MeHis1
His91
Tyr224 
N/D (starch) N/D (C1) Activity inferred from refs [70,71[72
AA13 Aspergillus oryzae AoAA13
AO090701000246
AOR_1_454114 
5T7N 3 mg/ml
0.02 M MES
pH 6.0
0.125 M NaCl 
0.02 M Zn-acetate
0.1 M Malate/MES/Tris pH 5.0
20% (w/v)
PEG3000 
P212121 1.60 Zn(II)
0.7 
MeHis1
His91
Tyr224 
N/D
(starch) 
N/D (C1) Glucosyl-maltotriose bound outside the active site.
Activity inferred from refs [70,71
[72
AA13 Aspergillus oryzae AoAA13
AO090701000246
AOR_1_454114 
5T7K 3 mg/ml
0.02 M MES
pH 6.0
0.125 M NaCl 
0.02 M Zn-acetate
0.1 M Malate/MES/Tris pH 5.0
20% (w/v)
PEG3000 
P212121 1.30 Zn(II)
0.2 
MeHis1
His91
Tyr224 
N/D (starch) N/D (C1) His91 flipped out of active site.
Stacking with Phe166.
Activity inferred from refs [70,71
[72
AA13 Aspergillus oryzae AoAA13
AO090701000246
AOR_1_454114 
5LSV 3 mg/ml
0.02 M MES
pH 6.0
0.125 M NaCl 
0.02 M Zn-acetate
0.1 M Malate/MES/Tris pH 5.0
20% (w/v)
PEG3000 
P212121 1.10 Zn(II)
A 0.8
B 0.2 
MeHis1
His91
Tyr224 
N/D (starch) N/D (C1) Maltose bound outside the active site.
Activity inferred from refs [70,71
[72
AA14 Pycnoporus coccineus PcAA14_B
PcAA14B 
5NO7 28 mg/ml
0.05 M Na-acetate pH 5.2 
2.4 M (NH4)2SO4 0.1 M citric acid
pH 4.4 
P422121 3.01 – His1
His99
Tyr176 
Xylan (cellulose-associated) C1 C1-oxidized xylo-oligosaccharides released after synergistic action with GH11 xylanases [21
AA15 Thermobia domestica TdAA15_A
TdAA15A 
5MSZ 28 mg/ml
0.05 M Na-acetate pH 5.2 
0.1 M Na-citrate
pH 5.5
0.1 M LiCl, 10–25% (w/v) PEG6000 
P22121 1.10 Cu(I) His1
His91
Tyr184 
Avicel β-chitin C1 First structure of an LPMO belonging to the phylum of Arthropoda [22

Full table is available at https://sid.erda.dk/sharelink/HB3w2p0k7s. Abbreviations: N/R: non reported; N/D: non determined.

Number of molecules in the asymmetric unit.

The criteria for assigning a Cu(II) or Cu(I) state were informed by structures where both states have been characterized [73]. The electron density of the equatorial exogenous ligand to the Cu (from weighed 2Fobs − Fcalc) should be more than 2 σ with more than 0.5 occupancy and distance to the copper less than 2.4 Å with similar criteria applying to the exogenous axial ligand, although with a distance of 2.8 Å. In structures where a distorted geometry is observed because of significant steric effects (most AA10), structures with a single exogenous ligand within 2.5 Å distance are taken as Cu(II). The occupancy of the metal is 1.00 if nothing else is indicated. If there is significant metal-site disorder with characteristics that could fit both states, the site is described as Cu(II)/Cu(I). When the copper occupancy was lower than 0.5, no oxidation state was assigned.

The overall IgG (immunoglobulin G)-like β-sandwich fold and His-brace (histidine-brace) motif [3] are shared by all LPMOs structurally investigated to date (Figure 2) including the two new families (AA14 and AA15). PcAA14_B [21] exhibits surface ripples formed by two loops, equivalent to the L2 and L3 loops of AA9s (Figure 3C). This gives the enzyme a contoured surface, which likely correlates with alternative substrate specificity compared with other LPMO families. In contrast, a flatter interaction surface is found in many AA9 LPMOs.

Representative structures for LPMO families.

Figure 2.
Representative structures for LPMO families.

Each LPMO family AA9–11 and AA13–15 exhibit the recognizable His-brace (top row, stick representation), composed of an N-terminal His (methylated in fungal LPMOs) and an internal His for the equatorial Cu-coordination positions. The His-brace is completed with a Tyr residue in the axial position, sometimes replaced with a Phe residue in some AA10 LPMOs (not shown). LPMO structures from all families have been solved with a Cu atom (orange spheres) in the active site, with the exception of the newly discovered AA14 family where a copper site similar to AA9 LPMOs was though confirmed by EPR spectroscopy [21]. LPMOs exhibit a common IgG-like fold (bottom row, cartoon representation) with variation in surface topology (grey) depending on loop regions and helices. The catalytic Cu-site is situated at the surface of the interaction interface. PDB codes of the representing structures are: 5ACH (AA9, yellow), 5OPF (AA10, magenta), 4MAI (AA11, purple), 4OPB (AA13, dark blue), 5NO7 (AA14, cyan) and 5MSZ (AA15, green).

Figure 2.
Representative structures for LPMO families.

Each LPMO family AA9–11 and AA13–15 exhibit the recognizable His-brace (top row, stick representation), composed of an N-terminal His (methylated in fungal LPMOs) and an internal His for the equatorial Cu-coordination positions. The His-brace is completed with a Tyr residue in the axial position, sometimes replaced with a Phe residue in some AA10 LPMOs (not shown). LPMO structures from all families have been solved with a Cu atom (orange spheres) in the active site, with the exception of the newly discovered AA14 family where a copper site similar to AA9 LPMOs was though confirmed by EPR spectroscopy [21]. LPMOs exhibit a common IgG-like fold (bottom row, cartoon representation) with variation in surface topology (grey) depending on loop regions and helices. The catalytic Cu-site is situated at the surface of the interaction interface. PDB codes of the representing structures are: 5ACH (AA9, yellow), 5OPF (AA10, magenta), 4MAI (AA11, purple), 4OPB (AA13, dark blue), 5NO7 (AA14, cyan) and 5MSZ (AA15, green).

Interactions with substrates and co-substrates.

Figure 3.
Interactions with substrates and co-substrates.

A and B illustrate oxygen species identified by advanced crystallographic studies, while C–E illustrate interactions with oligosaccharides. (A) 100 K resting-state X-ray (dark blue; PDB entry: 5TKG) and jointly refined room temperature neutron/X-ray structure (light blue; PDB entry: 5TKI) of NcAA9_D. In the X-ray 100 K structure, a dioxygen species has been modelled in two of the four molecules. The neutron scattering length density (SLD) map corresponding to the joint refinement is shown (2fo − fc at 1.5 σ contour level). (B) Neutron structure of JdAA10_A (PDB entry: 5VG1). In the shown neutron structure, an equatorial ligand (modelled as a peroxide ion) is only visible in one molecule of the asymmetric unit, while in the corresponding X-ray structure, equatorial dioxygen species are modelled in both molecules in the asymmetric unit (though in different orientations). Difference density at the N-terminus provides information of protonation states. The neutron SLD map (2fo − fc) is shown at 1.0 σ contour level, and difference density (fo − fc, green mesh) is at 3.0 σ contour level. Difference density at the modelled peroxide ion is 1.77 Å from the nearest oxygen atom. (C) Top view of LsAA9_A in complex with cellopentaose (yellow). Loops involved in substrate binding are highlighted in colour: LC (magenta); L2 (cyan); L3 (green); L8 (orange). (D) LsAA9_A in complex with xylopentaose. Binding of an axial oxygen mimic (Cl, yellow sphere) is allowed due to a difference in the conformation of substrate at the +1 subsite compared with LsAA9_A in complex with cello-oligosaccharides in which the oxygen mimic is bound in the equatorial position (as shown for cellotriose in (E)).

Figure 3.
Interactions with substrates and co-substrates.

A and B illustrate oxygen species identified by advanced crystallographic studies, while C–E illustrate interactions with oligosaccharides. (A) 100 K resting-state X-ray (dark blue; PDB entry: 5TKG) and jointly refined room temperature neutron/X-ray structure (light blue; PDB entry: 5TKI) of NcAA9_D. In the X-ray 100 K structure, a dioxygen species has been modelled in two of the four molecules. The neutron scattering length density (SLD) map corresponding to the joint refinement is shown (2fo − fc at 1.5 σ contour level). (B) Neutron structure of JdAA10_A (PDB entry: 5VG1). In the shown neutron structure, an equatorial ligand (modelled as a peroxide ion) is only visible in one molecule of the asymmetric unit, while in the corresponding X-ray structure, equatorial dioxygen species are modelled in both molecules in the asymmetric unit (though in different orientations). Difference density at the N-terminus provides information of protonation states. The neutron SLD map (2fo − fc) is shown at 1.0 σ contour level, and difference density (fo − fc, green mesh) is at 3.0 σ contour level. Difference density at the modelled peroxide ion is 1.77 Å from the nearest oxygen atom. (C) Top view of LsAA9_A in complex with cellopentaose (yellow). Loops involved in substrate binding are highlighted in colour: LC (magenta); L2 (cyan); L3 (green); L8 (orange). (D) LsAA9_A in complex with xylopentaose. Binding of an axial oxygen mimic (Cl, yellow sphere) is allowed due to a difference in the conformation of substrate at the +1 subsite compared with LsAA9_A in complex with cello-oligosaccharides in which the oxygen mimic is bound in the equatorial position (as shown for cellotriose in (E)).

The TdAA15_A structure, representing the other newly discovered family AA15 [22], contains two almost coplanar Tyr residues exposed on the surface on either side of the active site. This feature might be important for binding larger substrates and surface aromatic residues are also found in many AA9 LPMOs. The active site copper is in a slightly distorted type II conformation, as also confirmed by EPR (electron paramagnetic resonance) analysis, including as most LPMOs a Tyr ligand, and with an Ala residue in the solvent facing the axial coordination site, as AA10s. Chitin-specific AA15 LPMOs were also identified. Although no structures were presented, it is clear that, like strictly chitin-specific members of AA10, they have a Phe instead of a Tyr proximal to the His-brace.

The first LPMO neutron crystallography structures have been published recently. Compared with X-ray crystallography, hydrogen positions can be visualized after deuterium exchange and radiation damage (including metal photoreduction) is eliminated. Thus, neutron structures may also be determined without cryo-cooling at catalytically relevant temperatures. However, low neutron fluxes lead to overall poorer quality structures, not just in resolution but also in data completeness. Therefore, most neutron structures are determined in combination with X-ray structures through joint refinement [74].

A study of NcAA9_D was presented [62,75], including both neutron and high-resolution X-ray data. In a resting-state cryo-temperature X-ray structure, the authors identify a molecular oxygen pre-binding pocket lined by H157 and Q166 and adjacent to the active site (Figure 3A, PDB 5TKG) in both molecules of the asymmetric unit. In a different X-ray structure (PDB 5TKH) obtained from crystals treated with ascorbic acid prior to freezing, the authors also model oxygen prebound to one protein molecule, but tentatively model an equatorial peroxo species () in the other of the two molecules (ideally to be confirmed by neutron studies, as the density could arise from two alternate conformations of water). In the neutron structure at ambient temperature (jointly refined with X-ray data), water molecules are modelled coordinating the copper and the data clearly show hydrogen bonding and protonation states near the active site (Figure 3A, PDB 5TKI). In particular, the significance of the protonation state of H157 is discussed and computationally investigated (later followed up in ref. [27]), focusing on the role of H157 protonation state in molecular oxygen pre-binding.

In ref. [66], a 1.1 Å resolution room temperature X-ray structure and a corresponding neutron structure at 2.1 Å for JdAA10_A are reported, both obtained in the absence of reducing agents. In contrast with NcAA9_D, JdAA10_A does not have an equivalent of H157 to form an oxygen pre-binding pocket. The authors model instead a dioxygen species equatorial to the copper in the X-ray structure, where they find coordination distances consistent with primarily Cu(II), and a peroxide ion in the neutron structure (in one of the protein chains only and with rather different orientations compared with the X-ray structure) (Figure 3B). While these observations are interesting, they must be taken with some caution, given that in the X-ray structure the dioxygen density could arise from waters in different position (due to partial photoreduction) and the neutron structure is based on much less complete and lower resolution data (the overall completeness is only 76%). The N-terminal histidine amino group of JdAA10_A is interpreted by the authors as a mix of protonation states (both within and between different chains in the asymmetric unit). This could have important implications for the mechanism of LPMOs, also reviewed elsewhere [9], but again could be influenced by the limits in resolution and completeness of the neutron data. Although neutron structures are not routinely obtainable, they clearly can add mechanistically relevant information, especially if considered with caution and coupled with other studies.

Structure, function and stability

The earliest mutagenesis studies on LPMOs were carried out on the putative chitin-binding site of CBP21 (SmAA10_A [76]) and the metal-binding site of TtAA9_E [1], but these studies preceded an understanding of the oxidative nature of LPMOs. Since, detailed mutagenesis studies have been somewhat scarce until recently.

His-brace residues are known to be essential since the initial studies. The importance of a conserved Gln in the vicinity was also established in ref. [1]. The role of the equivalent Gln (Q167) and another conserved residue (H161) in the secondary copper coordination sphere was further probed in MtAA9_D [47]. These two residues are the same as suggested to pre-bind oxygen in NcAA9_D [62] (Figure 3A) and also discussed in ref. [27]. Most substitutions of Q167 and H161 reduced but did not abolish product formation from PASC (phosphoric-acid swollen cellulose) and O2 consumption, and one in particular, H161Q, showed strong uncoupling of lytic activity (reduced) from oxygen consumption (similar to wild type). Combining the results with EPR spectroscopy on the variants, the authors concluded that H161 and Q167 are both involved in O2 turnover, while Q167 also helps in tuning the electronic environment on the copper, likely through interaction with the Tyr occupying the axial coordination site [47].

Detailed sequence analysis [67] identified four residues (three in the L2 loop) in AA10 enzymes as potential determinants of regiospecificity. As often in protein engineering, alteration of product ratio was at the price of activity loss, but, as in a recent publication [77] on HjAA9_A, the study showed that residues likely to be involved in substrate positioning are key determinants of the C1 : C4 oxidation ratio, while the recent suggestion [78] that copper axial ligand accessibility was important was not supported by this study. Interestingly, both C4 oxidation and activity on chitin could be abolished by mutagenesis.

The association of LPMOs catalytic domains with carbohydrate-binding modules (CBMs) is common and its effect on activity has been investigated to some extent [69,79,80] as have the effect of surface aromatics on substrate binding and activity (e.g. [1,69]). Apart from direct and expected effects on substrate binding, CBM removal, though often applied for structural and sometimes functional characterization, has been reported to have adverse effect on LPMO stability [67]. Removal of CBM has not been shown to alter the C1 : C4 oxidation ratio [64,67].

The first structure of an LPMO-associated CBM was recently solved using NMR (nuclear magnetic resonance) and along with the crystal structure of ScAA10_C used to generate a structural model of the full-length enzyme [80].

The stability of NcAA9_C was investigated, in detail, by differential scanning fluorimetry [25]. pH below 6 has a negative impact on LPMO stability, which fits the His-brace disorder observed in a low pH LsAA9_A structure [60]. Reducing agents were destabilizing in the absence of carbohydrate substrates, an effect that could be counteracted by catalase or by substrate addition, underlying once more the deleterious effect of free H2O2. As shown previously biochemically (first in ref. [81]) and structurally for, for example, AA13, demetallation has adverse effects [72]. A 12°C increase in Tm was obtained recently for ScAA10 by engineering of two disulfide bridges [82].

Fungal LPMOs exhibit methylation of the N-terminal His (Figure 2). The role of this methylation was recently investigated through parallel assays for TaAA9_A expressed in Pichia (unmethylated) and Aspergillus (methylated) respectively, which suggested that the methylation is involved in protection against auto-oxidative inactivation of the LPMO [83].

As mentioned above, different binding modes have been observed for different substrates (e.g. cello- and xylo-oligosaccharides in ref. [15], see the next section), some leading to non-canonical degradation pathways. Such off-pathways may leave LPMOs more susceptible to auto-oxidative damage. In fact, recently some substrate-binding variants of SmAA10_A have been shown to have similar initial catalytic rates as wild type but been much less stable over time [84].

Interaction with substrates

One very important aspect of LPMO research has been devoted to characterize the substrate range of these enzymes. With the exception of the starch-active AA13 family, almost all LPMOs, to date, are reported to have activity on β-1,4-linked polymers of glucose (e.g. cellulose) or N-acetyl-glucosamine (chitin). Initially identified for their action on crystalline substrate, increasingly, LPMOs acting on soluble polysaccharides (e.g. β-glucans and xyloglucan [20,85]) and even oligosaccharides [35,36,86] have been identified. While the biological significance of LPMO acting on small/soluble substrates is unclear, such LPMOs are excellent model systems for kinetic studies and high-resolution crystallography [34,35].

The first structures of an LPMO–carbohydrate complex (LsAA9_A in complex with cello-oligosaccharides) were published in 2016 [35]. Together with spectroscopic and biochemical data, they helped elucidate the initial steps in this LPMO's reaction, showing that the axial water is displaced by the substrate and a Cl mimicking an oxygen species was bound equatorially instead of water.

In a follow-up study [15], LsAA9_A showed activity on several β-1,4-linked primarily glucose-backbone polysaccharides and in addition had low but significant activity on isolated xylan and xylo-oligosaccharides (other enzymes act on xylan as part of complex substrates [21,31]). CvAA9_A, a closely related enzyme, was found to lack three residues responsible for binding at the +2 site compared with LsAA9_A, explaining different product patterns. A series of complex structures showed a distinct binding mode for xylopentaose compared with cellopentaose and ligands with similar backbones (Figure 3D,E), which all displace the axial copper ligand. In contrast, xylopentaose allows binding of an axial ligand (here Cl). EPR spectra and different reductant dependence for action on xylan and PASC, in combination with the crystallographic evidence, suggest that the same LPMO can act through different mechanistic pathways, as already discussed above. The liganded structures of LsAA9_A are to date the only experimental high-resolution information on substrate–LPMO interaction (despite considerable reported efforts, for example, in refs [72,78]) and have been the starting point for several of the computational and biochemical studies referred to in this review, but details cannot be directly transferred to diverse LPMOs. Mapping of residues interacting with polysaccharides in the AA9 and AA10 families has been achieved by NMR spectroscopy [32,87] and, for AA9, was in qualitative agreement with the crystallographic results on short substrates. NMR spectroscopy has additionally been exploited to identify changes in complex substrates [21,88] on action by LPMOs. Higher affinity of substrate for the Cu(I) compared with Cu(II) form of two AA9 enzymes [25,34] has been reported.

The interactions of the C1-specific HiAA9_B with crystalline cellulose [59] have been probed by MD (molecular dynamics) simulations and found two stable binding modes, one in which three surface Tyr residues bind the same cellulose chain and one in which they bind three adjacent chains. The simulations suggest that an axial bound activated oxygen ligand could attack the C1 and support proximity as a major factor affecting regioselectivity [35,67], as previously suggested. A study of SmAA10_A binding to crystalline chitin-utilizing experimental constraints (mutagenesis data, product patterns and NMR spectroscopy [87,89]) together with QM/MM (quantum mechanics/molecular mechanics) and MD calculations reveals an inverted interaction orientation compared with LsAA9_A complex crystal structures [90]. Also in this case, the interaction suggested close proximity between the activated oxygen species and the oxidized C1 bond. A channel of ∼12 Å, gated by a Glu residue, could allow O2, H2O or H2O2 to access the Cu after docking onto the substrate.

Atomic resolution structures and studies with model substrates are important to understand the specificity and mechanism of LPMOs. However, their interaction with crystalline and complex substrates is more biologically and industrially relevant. In one of the first experimental LPMO single-molecules studies [91], it has been shown by microscopic techniques including atomic force microscopy (AFM) that an NcAA9_F is localized to crystalline regions of cellulose and thereby allows hydrolases to digest recalcitrant patches on the substrate. A more recent study by the same group [92] shows co-localization of NcAA9_C and NcAA9_F (one with C1 and one with C4 preference) on the crystalline surfaces also targeted by a synergistic cellobiohydrolase. In contrast with a recent AFM study of HjAA9_A on bacterial microcrystalline cellulose [93], where the LPMO showed a stop-and-go behavior, NcAA9_C and NcAA9_F appeared immobile. Using synchrotron UV (ultraviolet) fluorescence imaging [94], it could be shown that LPMOs act in synergy with cellulases to degrade miscanthus plant cell walls, and synchrotron infrared spectroscopy showed tissue-dependent effects. In terms of biotechnological applications, in line with [88], endoglucanase, LPMO and xylanases were shown to facilitate nanofibrillation of paper pulp [95], potentially reducing the need for mechanical refining while resulting in a pulp with a more uniform nanofibril composition. Moreover, it was recently demonstrated that LPMOs can be used to functionalize chitin and that such treatment reduces particle size but not crystallinity [96].

The biological context of LPMOs

LPMOs are widespread and abundant in filamentous fungi, but the biological role of the multiplicity of, for example, AA9 in fungal genomes (up to 50 genes) is still not understood. The major evolutionary constrain within each AA family appears to be factors different from substrate specificity.

Three AA9 LPMOs from the white-rot phytopathogenic fungus Heterobasidion irregulare have been studied biochemically [59,97]. This fungus causes wood decay in conifers, and this and other data [11] support a scenario where multiple AA9 LPMOs are required to mediate the decomposition of lignocellulose. In addition, the AA14s seem to be restricted to wood-degrading fungi where these xylan-specific LPMOs may target the recalcitrant xylan exposed on the surface of cellulose elementary fibres [21].

AA9 genes are also found in fungi that have mutualistic relationships with plants (e.g. mycorrhiza) [98100], but their biological function needs to be established. The biological diversity in LPMO-housing organisms keeps expanding. Functional AA9 LPMOs acting on cellulose and xyloglucan were found in the yeast Geotrichum candidum, which is well known from brie and camembert cheese [52].

LPMOs were thought to reside only in the world of microbes, but are now known also to be found in animals (AA15). AA15 is also found in oomycetes, multicellular and unicellular algae, Ichtyosporea and vira, but the biological role remains to be unravelled. The first biochemically characterized AA15s were isolated from Thermobia domestica, an insect which can digest crystalline cellulose without microbial assistance [22]. Contrary to T. domestica, the wood-boring shipworm Lyrodus peicellatus carry bacterial (AA10) LPMOs associated with their gills rather than endogenous LPMOs [101]. Such a symbiotic relationship is similar to the cellulolytic and aerobic strain of actinobacteria Streptomyces sp. SirexAA-E, which was isolated form the highly destructive pine-boring woodwasp Sirex noctilio. The genome of this Streptomyces strain encodes six AA10s of which three are specifically induced by cellulose [102,103].

In conclusion, the scientific community around LPMOs is learning with rapid pace and it will be important to keep a close eye on the development in coming years. How many LPMO families remain to be identified? Will they all share the same overall structure? Will the substrate range expand beyond polysaccharides? Do LPMOs all share a common mechanism or are there considerable variations? What are the roles of LPMOs in the animal kingdom? Can we pin-point the Cu-oxygen species directly involved with proton abstraction? These questions and many more remain open.

Abbreviations

     
  • AA

    auxiliary activity

  •  
  • AFM

    atomic force microscopy

  •  
  • CBM

    carbohydrate-binding module

  •  
  • CBP

    chitin-binding protein

  •  
  • EPR

    electron paramagnetic resonance

  •  
  • GH

    glycoside hydrolase

  •  
  • His-brace

    histidine-brace

  •  
  • IgG

    immunoglobulin G

  •  
  • LPMO

    lytic polysaccharide monooxygenase

  •  
  • MD

    molecular dynamics

  •  
  • NMR

    nuclear magnetic resonance

  •  
  • PASC

    phosphoric-acid swollen cellulose

  •  
  • SLD

    scattering length density

Funding

The authors' research on LPMOs is funded by the Novo Nordisk Foundation HOPE project [NNF17SA0027704] (L.L.L., K.S.J. and T.T.). K.E.H.F. thanks the Carlsberg Foundation for financial support through an Internationalisation Postdoc Fellowship [grant n°CF16-0673 and n°CF17-0533]. K.E.H.F. has received the support of the EU in the framework of the Marie-Curie FP7 COFUND People Programme, through the award of an AgreenSkills+ fellowship [under grant agreement n°609398]. L.L.L., T.T. and K.E.H.F. are members of ISBUC, Integrative Structural Biology at the University of Copenhagen (www.isbuc.ku.dk).

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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